Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity.

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets.

Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons.

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism.

Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters.

The inhibitory neurotransmitter gamma-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells.

Palmitoylation cycles and regulation of protein function (Review).

The efficacy and success of many cellular processes is dependent on a tight orchestration of proteins trafficking to and from their site(s) of action in a time-controlled fashion. Recently, a dynamic cycle of palmitoylation/de-palmitoylation has been shown to regulate shuttling of several proteins, including the small GTPases H-Ras and N-Ras, and the GABA-synthesizing enzyme GAD65, between the Golgi compartment and either the plasma membrane or synaptic vesicle membranes. These proteins are peripheral membrane proteins that in the depalmitoylated state cycle rapidly on and off the cytosolic face of ER/Golgi membranes.

A palmitoylation cycle dynamically regulates partitioning of the GABA-synthesizing enzyme GAD65 between ER-Golgi and post-Golgi membranes.

GAD65, the smaller isoform of the enzyme glutamic acid decarboxylase, synthesizes GABA for fine-tuning of inhibitory neurotransmission. GAD65 is synthesized as a soluble hydrophilic protein but undergoes a hydrophobic post-translational modification and becomes anchored to the cytosolic face of Golgi membranes. A second hydrophobic modification, palmitoylation of Cys30 and Cys45 in GAD65, is not required for the initial membrane anchoring but is crucial for post-Golgi trafficking of the protein to presynaptic clusters.